Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44704—Details; Accessories
  • G01N27/44747—Composition of gel or of carrier mixture
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/24—Extraction; Separation; Purification by electrochemical means
  • C07K1/26—Electrophoresis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44756—Apparatus specially adapted therefor
  • G01N27/44791—Microapparatus

Definitions

• aspects of the invention are generally directed to the field of capillary electrophoresis, in particular to microchip capillary electrophoresis.
• MCE has emerged as a promising technique with growing use in the pharmaceutical industry for characterizing biopharmaceuticals, quality control, and for drug discovery, it can be prone to assay interferences.
• MCE assays and reagents to assess purity and to identify impurities in protein drug product samples are provided.
• Methods for analyzing analytes in a protein drug sample are provided.
• Preferred protein drugs include but are not limited to recombinant proteins such as antibodies and antigen binding fragments thereof, and fusion proteins.
• the assays employ MCE techniques to separate, identify, and quantify protein product and impurities in the protein product.
• Impurities include, but are not limited to protein aggregates, protein fragments, protein multimers, and assay contaminants. Reducing and non-reducing buffers are also provided.
• the MCE assays and reagents provided herein may be used in the analysis and purity testing of Anti-SARS-CoV-2 products, such as therapeutic protein products including REGEN-COVTM (casirivimab and imdevimab).
• An embodiment provides a non-reducing aqueous electrophoresis sample buffer containing an alkylating agent such as 2-iodoacetamide (IAM), iodoacetic acid (IAA), or N- ethylmaleimide (NEM).
• an alkylating agent such as 2-iodoacetamide (IAM), iodoacetic acid (IAA), or N- ethylmaleimide (NEM).
• the non-reducing aqueous electrophoresis sample buffer contains an alkylating agent, 155 to 175 mM 2-iodoacetamide; 0.50 to 1.5% lithium dodecyl sulfate; and 65 to 95 mM sodium phosphate, wherein the aqueous electrophoresis sample buffer has a pH of less than 7. In an embodiment, the pH of the buffer is 6. In another embodiment, the aqueous buffer contains 166 mM 2-iodoacetamide, 0.81% lithium dodecy
• the non-reducing aqueous electrophoresis sample buffer contains an alkylating agent, for example 50 to 250 or 155 to 200 or 155 to 250 mM 2-iodoacetamide; 0.50 to 1.5% lithium dodecyl sulfate; and 40 to 80 mM or 50 to 70 mM sodium phosphate, wherein the aqueous electrophoresis sample buffer has a pH of less than or equal to 8.
• the pH of the buffer is 8.
• the aqueous buffer contains 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate.
• the non-reducing aqueous electrophoresis sample buffer contains an alkylating agent, for example 155 to 200 mM 2-iodoacetamide; 0.50 to 1.5% lithium dodecyl sulfate; and 50 to 70 mM sodium phosphate, wherein the aqueous electrophoresis sample buffer has a pH 8 or less.
• the pH of the buffer is 6.
• the aqueous buffer contains 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate.
• a reducing buffer is also provided.
• the reducing buffer is an aqueous electrophoresis sample buffer containing 0.5 to 1.5% lithium dodecyl sulfate, 55 to 85 mM sodium phosphate, and a reducing agent, wherein the aqueous electrophoresis sample buffer has a pH greater than 8.
• the pH of the buffer is 9.
• the reducing buffer contains 135 to 155 mM dithiothreitol.
• Still another embodiment provides a reducing buffer containing 0.69 % lithium dodecyl sulfate, 69 mM sodium phosphate, and 142 mM dithiothreitol.
• the reducing buffer is an aqueous electrophoresis sample buffer containing 0.5 to 1.5% lithium dodecyl sulfate, 45 to 85 mM sodium phosphate, and a reducing agent, wherein the aqueous electrophoresis sample buffer has a pH of 8 or greater.
• the pH of the buffer is 8.
• the reducing buffer contains 80 to 155 mM dithiothreitol.
• Still another embodiment provides a reducing buffer containing 1.2 % lithium dodecyl sulfate, 60 mM sodium phosphate, and 80 mM dithiothreitol.
• HEPES based buffers can also be used with the disclosed methods.
• One embodiment provides a non-reducing HEPES based aqueous electrophoresis sample buffer containing an alkylating agent, for example 55 to 75 mM 2-iodoacetamide; 0.1 to 1.0 % lithium dodecyl sulfate; 5 to 85 mM HEPES, and 5 to 115 mM sodium chloride, wherein the aqueous electrophoresis sample buffer has a pH of less than 9.
• the pH of the buffer is 8.
• the aqueous buffer contains 66.4 mM 2-iodoacetamide, 0.32% lithium dodecyl sulfate, 16.2 mM HEPES, and 48.6 mM sodium chloride.
• Another embodiment provides a reducing HEPES based aqueous electrophoresis sample buffer containing 0.05 to 0.75 % lithium dodecyl sulfate, 5 mM to 115 mM sodium chloride, 5 mM to 115 mM HEPES, and a reducing agent, wherein the aqueous electrophoresis sample buffer has a pH greater than 7.
• the pH of the buffer is 8.
• the reducing buffer contains 35 to 50 mM dithiothreitol.
• Still another embodiment provides a reducing buffer containing 0.28 % lithium dodecyl sulfate, 41.5 mM sodium chloride, 13.8 mM HEPES, and 42.5 mM dithiothreitol.
• One embodiment provides a non-reducing MCE method for identifying contaminants or impurities in a protein drug sample including the steps of adding the protein sample to a non reducing buffer discussed above to form a buffered protein drug sample.
• the buffered protein dmg sample is heated to between 65 to 85 °C for 5 to 15 minutes to form a denatured buffered protein drug sample.
• the buffered protein drug sample is heated to 70 °C for 10 min.
• a non-reducing MCE method for identifying contaminants or impurities in a protein drug sample includes the steps of adding the protein sample to a non reducing buffer discussed above to form a buffered protein drug sample.
• the buffered protein dmg sample is heated to between 50 to 72 °C for 5 to 15 minutes to form a denatured buffered protein dmg sample.
• the buffered protein dmg sample is heated to between 45 to 75 °C for 5 to 15 minutes to form a denatured buffered protein dmg sample.
• the buffered protein dmg sample is heated to 60 °C- 65 °C for 10 min.
• the buffered protein dmg sample is heated to 63 °C for 10 min.
• the buffered protein dmg sample is heated to 60 °C for 10 min.
• the protein dmg sample is mixed with a detectable label and heated at 30 to 40 °C for 20 to 40 minutes or for 10 to 40 minutes to form a denatured labeled protein dmg sample. In other embodiments, the protein dmg sample is mixed with a detectable label and heated at 30 to 40 °C for 15 minutes to form a denatured labeled protein dmg sample.
• a detectable label includes, but is not limited to Dyomics DY-631 NHS Ester.
• the label is diluted by MilliQ purified water, and in other embodiments the label is diluted in a sodium phosphate buffer.
• the detectable label is reconstituted with dimethyl sulfoxide (DMSO) followed by dilution with 200 mM Sodium Phosphate pH 7.2 buffer.
• DMSO dimethyl sulfoxide
• Other detectable labels can be used, including other dyes, fluorophores, chromophores, mass tags, quantum dots and the like and those disclosed in US Patent No. 6,924,372.
• the protein dmg sample with added label is heated to 35 °C for 30 minutes.
• the protein dmg sample with added label is heated to 35 °C for 15 minutes. Excess label is optionally removed from the sample, for example by using a spin filter. In still another embodiment, excess label is not quenched. In still another embodiment, excess label is not removed. [0018]
• the denatured labeled protein drug product is diluted and subjected to MCE to separate the diluted protein drug sample on a microchip capillary electrophoresis system to produce an electropherogram. In one embodiment the final concentration of a sample starting at 0.5 mg/ml that is then injected over the microchip is 9 pg/ml to MCE.
• the final concentration of a sample starting at 0.2 mg/ml that is then injected over the microchip is 3.6 pg/ml to MCE.
• the electropherogram contains peaks corresponding to the protein drug product and impurities. The method concludes by identifying peaks in the electropherogram corresponding to the protein drug product, contaminants, and/or impurities.
• Another embodiment provides a reducing MCE method for identifying contaminants or impurities in a protein drug sample.
• the method begins by adding the protein sample to any one of the reducing buffers described above to form a buffered protein drug sample.
• the buffered protein drug sample is denatured by heating the buffered protein drug sample to 65 to 85 °C, preferably to 75 °C for 10 minutes to form a denatured protein drug sample.
• the protein drug sample is mixed with a detectable label and heated at 30 to 40 °C for 20 to 40 minutes to form a denatured labeled protein drug sample.
• the protein drug sample with added label is heated to 35 °C for 30 minutes.
• the buffered protein drug sample is denatured by heating the buffered protein drug sample to 50 - 72 °C, preferably to 60 °C for 10 minutes to form a denatured protein drug sample.
• the protein drug sample is mixed with a detectable label and heated at 30 to 40 °C for 15 minutes to form a denatured labeled protein drug sample.
• the protein drug sample with added label is heated to 35 °C for 15 minutes.
• Excess label is optionally removed from the sample, for example by using a spin filter. In still another embodiment, excess label is not quenched. In still another embodiment, excess label is not removed.
• An exemplary detectable label includes, but is not limited to Dyomics DY-631 NHS Ester. Other detectable labels that can be used include other dyes, fluorophores, chromophores, mass tags, quantum dots and the like and those disclosed in US Patent No. 6,924,372.
• the established assay range for sample concentration is from 0.4 mg/ml to 0.6 mg/ml, corresponding to a final concentration being analyzed of about 7 pg/ml to 11 mg/ml which is subjected to MCE analysis on a microchip capillary electrophoresis system to produce an electropherogram.
• the established assay range for sample concentration is from 0.2 mg/ml to 0.6 mg/ml, corresponding to a final concentration being analyzed of about 3.6 pg/ml to 11 pg/ml. The method concludes by identifying peaks in the electropherogram corresponding to the protein drug product, contaminants, and/or impurities.
• Figure 1A shows an electropherogram of a typical non-reduced sample analysis.
• Figure IB shows an electropherogram of a typical reduced sample analysis.
• the X-axis represents time in minutes, and the Y-axis represents the relative fluorescence units (RFU). Increased migration time corresponds to increased protein size.
• REU relative fluorescence units
• Protein refers to a molecule comprising two or more amino acid residues joined to each other by a peptide bond. Protein includes polypeptides and peptides and may also include modifications such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation and ADP-ribosylation. Proteins can be of scientific or commercial interest, including protein-based drugs, and proteins include, among other things, enzymes, ligands, receptors, antibodies and chimeric or fusion proteins.
• Proteins are produced by various types of recombinant cells using well-known cell culture methods, and are generally introduced into the cell by genetic engineering techniques (e.g., such as a sequence encoding a chimeric protein, or a codon-optimized sequence, an intronless sequence, etc.) where it may reside as an episome or be integrated into the genome of the cell.
• genetic engineering techniques e.g., such as a sequence encoding a chimeric protein, or a codon-optimized sequence, an intronless sequence, etc.
• Antibody refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
• Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region.
• the heavy chain constant region contains three domains, CHI, CH2 and CH3.
• Each light chain has a light chain variable region and a light chain constant region.
• the light chain constant region consists of one domain (CL).
• the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
• CDR complementarity determining regions
• Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
• the term “antibody” includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass.
• the term “antibody” includes antibody molecules prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell transfected to express the antibody.
• the term antibody also includes bispecific antibody, which includes a heterotetrameric immunoglobulin that can bind to more than one different epitope. Bispecific antibodies are generally described in US Patent No. 8,586,713.
• Fc fusion proteins comprise part or all of two or more proteins, one of which is an Fc portion of an immunoglobulin molecule, which are not otherwise found together in nature.
• Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et ah, Proc. Natl. Acad. Sci USA, 88: 10535 (1991); Byrn et al., Nature 344:677 (1990); and Hollenbaugh et al., “Construction of Immunoglobulin Fusion Proteins”, in Current Protocols in Immunology, Suppl.
• Receptor Fc fusion proteins comprise one or more extracellular domain(s) of a receptor coupled to an Fc moiety, which in some embodiments comprises a hinge region followed by a CH2 and CH3 domain of an immunoglobulin.
• the Fc-fusion protein comprises two or more distinct receptor chains that bind to a one or more ligand(s).
• an Fc-fusion protein is a trap, such as for example an IL-1 trap or VEGF trap.
• MCE Microchip Capillary Electrophoresis
• CE capillary electrophoresis
• Protein drugs include, but are not limited to antibodies and antigen binding fragments thereof, fusion proteins, and recombinant proteins.
• the assays employ MCE techniques to separate, identify, and quantify protein product and impurities in the protein product.
• Impurities include, but are not limited to protein aggregates, protein fragments, protein multimers, and assay contaminants. Reducing and non-reducing buffers are also provided.
• Microchip Capillary Electrophoresis provides analysis of protein purity and impurities suitable for in-process testing, lot release, and stability indication.
• Protein samples such as casirivimab and imdevimab, were prepared by denaturing the sample through heating and addition of lithium dodecyl sulfate (LDS) with either an alkylating agent (non-reduced) or a reducing agent (reduced). Not intending to be bound by theory, this produces linear negatively charged protein-LDS polypeptide chains, which are then incubated with an amine ester dye to covalently label any free amine groups.
• LDS lithium dodecyl sulfate
• Labeled samples may be injected onto a microchip where the application of a current separates the sample components according to mass to charge ratio. Detection can be performed by laser-induced fluorescence, producing an electropherogram. Analysis of the electropherogram provides the relative purity of the sample compared to the impurities present.
• One embodiment provides a non-reducing aqueous electrophoresis sample buffer containing 155 to 200 mM of an alkylating agent for example 2-iodoacetamide; 0.50 to 1.5% lithium dodecyl sulfate; and 60 to 95 mM sodium phosphate, wherein the aqueous electrophoresis sample buffer has a pH of less than 7.
• the pH of the buffer is 6.
• the aqueous buffer contains 166 mM 2-iodoacetamide, 0.81% lithium dodecyl sulfate, and 81 mM sodium phosphate.
• Another embodiment provides a non-reducing aqueous electrophoresis sample buffer containing 155 to 200 mM of an alkylating agent; 0.50 to 1.5% lithium dodecyl sulfate; and 60 to 95 mM sodium phosphate, wherein the aqueous electrophoresis sample buffer has a pH of less than or equal to 8.
• the pH of the buffer is 8.
• the aqueous buffer contains 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate.
• Another embodiment provides a non-reducing aqueous electrophoresis sample buffer containing 155 to 200 mM of an alkylating agent; 0.50 to 1.5% lithium dodecyl sulfate; and 60 to 95 mM sodium phosphate, wherein the aqueous electrophoresis sample buffer has a pH of less than 8.
• the pH of the buffer is 6.
• the aqueous buffer contains 200 mM 2-iodoacetamide, 1.2% lithium dodecyl sulfate, and 60 mM sodium phosphate.
• a reducing buffer is also provided.
• the reducing buffer is an aqueous electrophoresis sample buffer containing 0.5 to 1.5% lithium dodecyl sulfate, 65 to 95 mM sodium phosphate, and a reducing agent, wherein the aqueous electrophoresis sample buffer has a pH greater than 8.
• the reducing buffer contains 0.5 to 1.5% lithium dodecyl sulfate, 45 to 95 mM sodium phosphate, and a reducing agent, and the aqueous electrophoresis sample buffer has a pH of 8 or greater.
• the pH of the buffer is 9.
• the pH of the buffer is 8.
• Reducing agents are known in the art.
• Exemplary reducing agents include but are not limited to dithiothreitol (DTT, CAS 3483-12-3), beta-mercaptoethanol (BME, 2BME, 2-ME, b- mer, CAS 60-24-2), 2-aminoethanethiol (2-MEA-HCl, also called cysteamine-HCl, CAS 156- 57-0), Tris (2-carboxyethyl) phosphine hydrochloride, (TCEP, CAS 5961-85-3), cysteine hydrochloride (Cys-HCl, CAS 52-89-1), or 2-mercaptoethanesulfonic acid sodium salt (MESNA).
• DTT dithiothreitol
• BME beta-mercaptoethanol
• 2-aminoethanethiol also called cysteamine-HCl, CAS 156- 57-0
• the reducing buffer contains 135 to 155 mM dithiothreitol. In another embodiment, the reducing buffer contains 80 to 155 mM dithiothreitol.
• Still another embodiment provides a reducing buffer containing 0.69 % lithium dodecyl sulfate, 69 mM sodium phosphate, and 142 mM dithiothreitol, or 1.2 % lithium dodecyl sulfate, 60 mM sodium phosphate, and 80 mM dithiothreitol.
• HEPES based buffers can also be used with the disclosed methods.
• One embodiment provides a non-reducing HEPES based aqueous electrophoresis sample buffer containing an alkylating agent, for example 55 to 75 mM 2-iodoacetamide; 0.1 to 1.0 % lithium dodecyl sulfate; 5 to 85 mM HEPES, and 5 to 115 mM sodium chloride, wherein the aqueous electrophoresis sample buffer has a pH of less than 9.
• the pH of the buffer is 8.
• the aqueous buffer contains 66.4 mM 2-iodoacetamide, 0.32% lithium dodecyl sulfate, 16.2 mM HEPES, and 48.6 mM sodium chloride.
• Another embodiment provides a reducing HEPES based aqueous electrophoresis sample buffer containing 0.05 to 0.75 % lithium dodecyl sulfate, 5 mM to 115 mM sodium chloride, 5 mM to 115 mM HEPES, and a reducing agent, wherein the aqueous electrophoresis sample buffer has a pH greater than 7.
• the pH of the buffer is 8.
• the reducing buffer contains 35 to 50 mM dithiothreitol.
• Still another embodiment provides a reducing buffer containing 0.28 % lithium dodecyl sulfate, 41.5 mM sodium chloride, 13.8 mM HEPES, and 42.5 mM dithiothreitol.
• One embodiment provides a non-reducing MCE method for identifying contaminants or impurities in a protein drug sample, the method including the steps of adding the protein sample to a non-reducing buffer discussed above to form a buffered protein drug sample.
• the buffered protein drug sample is heated to between 50 to 85 °C for 5 to 15 minutes to form a denatured buffered protein drug sample.
• the buffered protein drug sample is heated to 75 °C for 10 min.
• the buffered protein drug sample is heated to between 45 to 75 °C for 5 to 15 minutes to form a denatured buffered protein drug sample.
• the buffered protein drug sample is heated to 60 °C for 10 min.
• the buffered protein drug sample is heated to 63 °C for 10 min.
• a detectable label is prepared to vendor recommendations in dimethyl sulfoxide (DMSO). Subsequent dilutions are prepared per vendor recommendations in MilliQ.
• the label is prepared by dilution in 200 mM sodium phosphate pH 7.2. In some embodiments, the label is diluted to a 5 m M solution. In other embodiments, the label is diluted to a 16 mM solution. The diluted label is then added to the denatured buffered protein drug sample and heated at 30 to 40 °C for 10 to 40 minutes to form a denatured labeled protein drug sample.
• a detectable label is then added to the denatured buffered protein drug sample and heated at 30 to 40 °C for 15 minutes to form a denatured labeled protein dmg sample.
• the denatured protein drug sample with added label is heated to 35 °C for 30 minutes.
• the denatured protein drug sample with added label is heated to 35 °C for 15 minutes.
• Excess label is optionally removed from the sample, for example by using a spin filter.
• excess label is not quenched.
• excess label is not removed.
• a preferred detectable label includes, but is not limited to Dyomics DY-631 NHS Ester.
• Other detectable labels can be used include other dyes, fluorophores, chromophores, mass tags, quantum dots and the like and those disclosed in US Patent No. 6,924,372.
• the denatured labeled protein drug product is diluted and subjected to MCE to separate the diluted protein drug sample on a microchip capillary electrophoresis system to produce an electropherogram.
• the final concentration of a sample starting at 0.5 mg/ml that is then injected over the microchip is 9 pg/ml to MCE.
• the sample starting concentration is 0.2 mg/ml.
• the electropherogram contains peaks corresponding to the protein drug product and impurities. The method concludes by identifying peaks in the electropherogram corresponding to contaminants or impurities.
• Another embodiment provides a reducing MCE method for identifying contaminants or impurities in a protein drug sample.
• the method begins by adding the protein drug sample to any one of the reducing buffers described above to form a buffered protein drug sample.
• the buffered protein drug sample is denatured by heating the buffered protein drug sample to 65 to 85 °C, preferably to 75 °C for 10 minutes to form a denatured protein drug sample.
• the buffered protein drug sample is denatured by heating the buffered protein drug sample to 50 to 72 °C, or 45 to 75 °C, or preferably to 60 °C for 10 minutes to form a denatured protein drug sample.
• the buffered protein drug sample is heated to 63 °C for 10 min. The temperature needed to denature the target protein drug sample may be changed according to the structure of the target protein drug sample.
• the protein drug sample with added label is then heated at 30 to 40 °C for 20 to 40 minutes or for 10 to 20 or for 10 to 40 minutes to form a denatured labeled protein drug sample.
• the protein drug product sample with added label is heated to 35 °C for 30 minutes.
• the protein drug product sample with added label is heated to 35 °C for 15 minutes.
• Excess label is optionally removed from the sample, for example by using a spin filter.
• excess label is not quenched.
• excess label is not removed.
• a preferred detectable label includes, but is not limited to Dyomics DY -631 NHS Ester.
• Other detectable labels that can be used include other dyes, fluorophores, chromophores, mass tags, quantum dots and the like and those disclosed in US Patent No. 6,924,372.
• the established assay range for sample concentration is from 0.4 mg/ml to 0.6 mg/ml, corresponding to a final concentration being analyzed of about 7pg/ml to 1 lpg/ml which is subjected to MCE analysis on a microchip capillary electrophoresis system to produce an electropherogram.
• the established assay range for sample concentration is from 0.2 mg/ml to 0.6 mg/ml, corresponding to a final concentration being analyzed of about 3.6 mg/ml to 11 mg/ml which is subjected to MCE analysis on a microchip capillary electrophoresis system to produce an electropherogram.
• the method concludes by identifying peaks in the electropherogram corresponding to protein drug product, contaminants, and/or impurities.
• Instrumentation for conducting the disclosed MCE assays is commercially available.
• the disclosed MCE assays are performed using LabChip GXII or LabChip GXII Touch HT and LabChip® HT Protein Express Chip.
• the protein of interest for example a protein drug product, assayed with the disclosed MCE assays and reagents can be any protein of interest suitable for expression in prokaryotic or eukaryotic cells and can be used in the engineered host cell systems provided.
• the protein of interest includes, but is not limited to, an antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, an ScFv or fragment thereof, an Fc-fusion protein or fragment thereof, a growth factor or a fragment thereof, a cytokine or a fragment thereof, or an extracellular domain of a cell surface receptor or a fragment thereof.
• Proteins of interest may be simple polypeptides consisting of a single subunit, or complex multisubunit proteins comprising two or more subunits.
• the protein of interest may be a biopharmaceutical product, food additive or preservative, or any protein product subject to purification and quality standards.
• the protein drug product is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen binding antibody fragment, a single chain antibody, a diabody, triabody or tetrabody, a Fab fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.
• the antibody is an IgGl antibody.
• the antibody is an IgG2 antibody.
• the antibody is an IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2/IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2/IgGl antibody. In one embodiment, the antibody is a chimeric IgG2/IgGl/IgG4 antibody. [0053] Embodiments can be used with any known therapeutic antibody therapeutic. In some embodiments, the antibody can be selected from the group consisting of an anti-Programmed Cell Death 1 antibody (e.g. an anti-PDl antibody as described in U.S. Pat. Appln. Pub. No.
• an anti-Programmed Cell Death 1 antibody e.g. an anti-PDl antibody as described in U.S. Pat. Appln. Pub. No.
• an anti-Programmed Cell Death Ligand-1 e.g., an anti-PD-Ll antibody as described in in U.S. Pat. Appln. Pub. No. US2015/0203580A1
• an anti-D114 antibody e.g., an anti- Angiopoetin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Pat. No. 9,402,898)
• an anti- Angiopoetin-Like 3 antibody e.g., an anti-AngPtl3 antibody as described in U.S. Pat.
• an anti-platelet derived growth factor receptor antibody e.g., an anti-PDGFR antibody as described in U.S. Pat. No. 9,265,827
• an anti-Erb3 antibody e.g., an anti-Prolactin Receptor antibody (e.g., anti-PRLR antibody as described in U.S. Pat. No. 9,302,015)
• an anti- Complement 5 antibody e.g., an anti-C5 antibody as described in U.S. Pat. Appln. Pub. No US2015/0313194A1
• an anti-TNF antibody e.g., an anti-epidermal growth factor receptor antibody
• an anti-EGFR antibody as described in U.S. Pat. No.
• an anti-Proprotein Convertase Subtilisin Kexin-9 antibody e.g., an anti-PCSK9 antibody as described in U.S. Pat. No. 8,062,640 or U.S. Pat. No. 9,540,449
• an Anti-Growth and Differentiation Factor-8 antibody e.g. an anti-GDF8 antibody, also known as anti-myostatin antibody, as described in U.S. Pat Nos. 8,871,209 or 9,260,515)
• an anti-Glucagon Receptor e.g. anti-GCGR antibody as described in U.S. Pat.
• an anti-VEGF antibody an anti-ILlR antibody
• an interleukin 4 receptor antibody e.g., an anti-IL4R antibody as described in U.S. Pat. Appln. Pub. No. US2014/0271681A1 or U.S. Pat Nos. 8,735,095 or 8,945,559
• an anti-interleukin 6 receptor antibody e.g., an anti-IL6R antibody as described in U.S. Pat. Nos.
• an anti-ILl antibody an anti-IL2 antibody, an anti-IL3 antibody, an anti-IL4 antibody, an anti-IL5 antibody, an anti-IL6 antibody, an anti-IL7 antibody, an anti-interleukin 33 (e.g., anti- IL33 antibody as described in U.S. Pat. Nos. 9,453,072 or 9,637,535), an anti-Respiratory syncytial virus antibody (e.g., anti-RSV antibody as described in U.S. Pat. Appln. Pub. No. 9,447,173), an anti-Cluster of differentiation 3 (e.g., an anti-CD3 antibody, as described in U.S. Pat.
• an anti- Cluster of differentiation 20 e.g., an anti-CD20 antibody as described in U.S. Pat. Nos. 9,657,102 and US20150266966A1, and in U.S. Pat. No. 7,879,984
• an anti-CD19 antibody e.g., an anti-CD28 antibody
• an anti- Cluster of Differentiation-48 e.g. anti-CD48 antibody as described in U.S. Pat. No. 9,228,014
• an anti-Fel dl antibody e.g. as described in U.S. Pat. No.
• an anti-Middle East Respiratory Syndrome virus e.g. an anti-MERS antibody as described in U.S. Pat. Appln. Pub. No. US2015/0337029A1
• an anti- Ebola virus antibody e.g. as described in U.S. Pat. Appln. Pub. No. US2016/0215040
• an anti- Zika virus antibody e.g. an anti-LAG3 antibody, or an anti-CD223 antibody
• an anti-Nerve Growth Factor antibody e.g. an anti-NGF antibody as described in U.S. Pat. Appln. Pub. No. US2016/0017029 and U.S. Pat. Nos.
• the bispecific antibody can be selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody (as described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and US20150266966A1), an anti-CD3 x anti-Mucin 16 bispecific antibody (e.g., an anti-CD3 x anti- Mucl6 bispecific antibody), and an anti-CD3 x anti- Prostate-specific membrane antigen bispecific antibody (e.g., an anti-CD3 x anti-PSMA bispecific antibody).
• an anti-CD3 x anti-CD20 bispecific antibody as described in U.S. Pat. Appln. Pub. Nos. US2014/0088295A1 and US20150266966A1
• an anti-CD3 x anti-Mucin 16 bispecific antibody e.g., an anti-CD3 x anti- Mucl6 bispecific antibody
• the protein of interest can be selected from the group consisting of casirivimab, imdevimab, ravulizumab-cwvz, abciximab, adalimumab, adalimumab-atto, ado-trastuzumab, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, brodalumab, canakinumab, capromab pendetide, certolizumab pegol, cemiplimab, cetuximab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzum
• the protein of interest is a recombinant protein that contains an Fc moiety and another domain, (e.g., an Fc-fusion protein).
• an Fc-fusion protein is a receptor Fc-fusion protein, which contains one or more extracellular domain(s) of a receptor coupled to an Fc moiety.
• the Fc moiety comprises a hinge region followed by a CH2 and CH3 domain of an IgG.
• the receptor Fc- fusion protein contains two or more distinct receptor chains that bind to either a single ligand or multiple ligands.
• an Fc-fusion protein is a TRAP protein, such as an IL-1 trap (e.g., rilonacept, which contains the IL-lRAcP ligand binding region fused to the I1-1R1 extracellular region fused to Fc of hlgGl; see U.S. Pat. No. 6,927,044, or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which comprises the Ig domain 2 of the VEGF receptor Fltl fused to the Ig domain 3 of the VEGF receptor Flkl fused to Fc of hlgGl; see U.S. Pat. Nos. 7,087,411 and 7,279,159).
• IL-1 trap e.g., rilonacept, which contains the IL-lRAcP ligand binding region fused to the I1-1R1 extracellular region fused to Fc of hlgGl
• a VEGF trap e.
• an Fc-fusion protein is a ScFv-Fc-fusion protein, which contains one or more of one or more antigen-binding domain(s), such as a variable heavy chain fragment and a variable light chain fragment, of an antibody coupled to an Fc moiety.
• the protein drug product assayed with the disclosed MCE assays and reagents are produced cell cultures.
• the cell cultures can be a "fed-batch cell culture” or “fed-batch culture” which refers to a batch culture wherein the cells and culture medium are supplied to the culturing vessel initially and additional culture nutrients are slowly fed, in discrete increments, to the culture during culturing, with or without periodic cell and/or product harvest before termination of culture.
• Fed-batch culture includes “semi-continuous fed-batch culture” wherein periodically whole culture (which may include cells and medium) is removed and replaced by fresh medium.
• Fed-batch culture is distinguished from simple "batch culture” whereas all components for cell culturing (including the animal cells and all culture nutrients) are supplied to the culturing vessel at the start of the culturing process in batch culture.
• Fed-batch culture may be different from “perfusion culture” insofar as the supernatant is not removed from the culturing vessel during a standard fed-batch process, whereas in perfusion culturing, the cells are restrained in the culture by, e.g., filtration, and the culture medium is continuously or intermittently introduced and removed from the culturing vessel.
• perfusion culture insofar as the supernatant is not removed from the culturing vessel during a standard fed-batch process
• perfusion culturing the cells are restrained in the culture by, e.g., filtration, and the culture medium is continuously or intermittently introduced and removed from the culturing vessel.
• removal of samples for testing purposes during fed-batch cell culture is contemplated.
• the cell culture can be a “continuous cell culture” which is a technique used to grow cells continually, usually in a particular growth phase. For example, if a constant supply of cells is required, or the production of a particular protein of interest is required, the cell culture may require maintenance in a particular phase of growth. Thus, the conditions must be continually monitored and adjusted accordingly in order to maintain the cells in that particular phase.
• the cells are cultured in cell culture medium.
• cell culture medium and “culture medium” refer to a nutrient solution used for growing mammalian cells that typically provides the necessary nutrients to enhance growth of the cells, such as a carbohydrate energy source, essential (e.g., phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine) and nonessential (e.g., alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine) amino acids, trace elements, energy sources, lipids, vitamins, etc.
• essential e.g., phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine
• nonessential e.g., alanine,
• Cell culture medium may contain extracts, e.g., serum or peptones (hydrolysates), which supply raw materials that support cell growth.
• Media may contain yeast-derived or soy extracts, instead of animal-derived extracts.
• Chemically defined medium refers to a cell culture medium in which all of the chemical components are known (i.e., have a known chemical structure). Chemically defined medium is entirely free of animal-derived components, such as serum- or animal-derived peptones. In one embodiment, the medium is a chemically defined medium.
• the solution may also contain components that enhance growth and/or survival above the minimal rate, including hormones and growth factors.
• the solution may be formulated to a pH and salt concentration optimal for survival and proliferation of the particular cell being cultured.
• a “cell line” refers to a cell or cells that are derived from a particular lineage through serial passaging or subculturing of cells. The term “cells” is used interchangeably with “cell population”.
• cell includes any cell that is suitable for expressing a recombinant nucleic acid sequence.
• Cells include those of prokaryotes and eukaryotes, such as bacterial cells, mammalian cells, human cells, non-human animal cells, avian cells, insect cells, yeast cells, or cell fusions such as, for example, hybridomas or quadromas.
• the cell is a human, monkey, ape, hamster, rat or mouse cell.
• the cell can be selected from the following cells: Chinese Hamster Ovary (CHO) (e.g., CHO Kl, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, lymphocyte, e.g., Jurkat (T lymphocyte) or Daudi (B lymphocyte), A431 (epidermal), U937,
• CHO Chinese Hamster Ovary
• COS e.g., COS-7
• retinal cell Vero
• CV1 e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21
• HeLa HepG2, WI38
• MRC 5 Colo25
• HB 8065 e.g., Jur
• the cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER.C6® cell).
• the cell is a CHO cell. In other embodiments, the cell is a CHO K1 cell.
• kits including the one or more of the disclosed buffers or ingredients to make the disclosed buffers.
• the kit can include a container for the buffers or ingredients.
• the buffers can be in solution or in lyophilized form.
• the kit optionally also includes a second container containing a diluent or reconstituting solution for the lyophilized formulation; and optionally, instructions for the use of the solution or the reconstitution and/or use of the lyophilized buffers or powdered ingredients.
• the kit may further include additional reagents needed to perform the disclosed MCE assays including one or more of a buffer, a diluent, and a filter.
• the buffer and reagents can be in a bottle, a vial, or test tube.
• Example 1 MCE assay for purity and impurity analysis of therapeutic proteins.
• Microchip Capillary Electrophoresis provides analysis of protein purity and impurities suitable for in-process testing, lot release, and stability indication.
• Protein samples of casirivimab and imdevimab were prepared by denaturing the sample through heating and addition of lithium dodecyl sulfate (LDS) with either an alkylating (non-reduced) or reducing agent (reduced). The resulting samples were then incubated with an amine ester dye to covalently label any free amine groups. Labeled samples were injected onto a microchip where the application of a current separated the sample components according to size (from low to high molecular weight). Detection was performed by laser-induced fluorescence, producing an electropherogram. Analysis of the electropherogram provided the relative purity of the sample compared to the impurities present.
• LDS lithium dodecyl sulfate
• amine ester dye to covalently label any free amine groups.
• Labeled samples were injected onto a
• Table 1 shows the workflow procedure for preparing a sample for a first MCE assay. Briefly, protein samples were diluted to 0.5 mg/ml. 1 m ⁇ of either non-reducing (NR) or reducing (R) denaturing buffer and 4 m ⁇ of the diluted sample were added to a 96-well plate. The sample was mixed, centrifuged, and heated for 10 minutes at the temperature specified for the product, typically 75°C. The samples were then labeled with 5 mM commercially available dye (for example Dyomics DY-631 NHS Ester, also called PICO dye). The samples were mixed, centrifuged, and then heated at 35°C for 30 minutes. The labeled sample was then diluted with 105 m ⁇ of dilute stop solution. The samples were separated using LabChip GXII or LabChip GXII Touch HT.
• NR non-reducing
• R reducing
• Table 1 Sample preparation method for a first MCE assay.
• Table 2 shows the workflow procedure for preparing a sample for a second MCE assay. Briefly, protein samples were diluted to 0.5 or 2 mg/ml. 10 m ⁇ of either non-reducing (NR) or reducing (R) denaturing buffer and 40 m ⁇ of the diluted sample were added to a 96-well plate.
• NR non-reducing
• R reducing
• the sample was mixed, centrifuged, and heated for 10 minutes at the temperature specified for the product, typically 60°C.
• the samples were then labeled with 16 mM commercially available dye (for example Dyomics DY-631 NHS Ester).
• the samples were mixed, centrifuged, and then heated at 35°C for 15 minutes.
• the labeled sample was then diluted with 105 m ⁇ of dilute stop solution or dilute solution.
• the samples were separated using LabChip GXII or LabChip GXII Touch HT.
• Alternate buffers were made using stock solutions and Milli-Q water include solutions of 100 mM Sodium Phosphate 2 % LDS pH 6 and 100 mM Sodium Phosphate 2% LDS pH 8. [0073] A non-reducing buffer was prepared by adding 34 m L 1M Iodoacetamide (IAM)
• An alternate non-reducing buffer was prepared by adding 800 pL 1M Iodoacetamide (IAM) (prepared fresh in Milli-Q® water) + 1200 m L 100 mM Sodium Phosphate 2% LDS pH 8. The final concentrations were 200 mM 2-iodoacetamide, 1.2 % lithium dodecyl sulfate, and 60 mM Sodium phosphate.
• Yet another non-reducing buffer was prepared by adding 800 pL 1M Iodoacetamide (IAM) (prepared fresh in Milli-Q® water) + 1200 m L 100 mM Sodium Phosphate 2% LDS pH 6. The final concentrations were 200 mM 2-iodoacetamide, 1.2 % lithium dodecyl sulfate, and 60 mM Sodium phosphate.
• a reducing buffer was prepared by adding 68 pL lOx Reducing agent (500 mM dithiothreitol (DTT) + 166 pL 100 mM Sodium Phosphate 1% LDS pH 9 + 6 pL Milli-Q® water. The final concentrations were 0.69% lithium dodecyl sulfate; 69 mM sodium phosphate, and 142 mM dithiothreitol.
• DTT dithiothreitol
• An alternate reducing buffer was prepared by adding 320 pL lOx Reducing agent (500 mM dithiothreitol (DTT) + 1200 pL 100 mM Sodium Phosphate 2% LDS pH 8 + 480 pL Milli- Q® water. The final concentrations were 1.2% lithium dodecyl sulfate; 60 mM sodium phosphate, and 80 mM dithiothreitol.
• DTT dithiothreitol
• Example 2C Non-reducing buffer, non-quenching
• the sample was heated at 63°C for 10 minutes.
• the Sample Labeling step used 16 m M dye solution.
• the Final Dilution step used 105 pL Dilute Solution.
• Example 2D Reducing buffer, non-auenching
• Microchip Capillary Electrophoresis allows for dramatically reduced sample analysis times, while maintaining the performance and reproducibility standards required for QC analysis.
• An MCE assay was developed using the non-reduced and reduced denaturing buffers disclosed herein.
• Figures 1A-1B show representative electropherograms of protein in non- reduced samples and reduced samples from the first MCE assay.
• Figure 1A shows a non-reduced analysis electropherogram.
• the main peak (MP) at 0.426 corresponds to the main antibody
• the labeled low molecular weight (LMW) peaks corresponds to antibody fragments.
• a peak corresponding to one or more high molecular weight fragments are present.
• Figure IB shows a reduced analysis electropherogram. Peaks corresponding to light chain (LC), heavy chain, and non-glycosylated heavy chain (NGHC), low molecular weight (LMW), and high molecular weight (HMW) antibody fragments are labeled accordingly. It should be noted that different products may have variation in the electropherograms produced, and therefore different labeling conventions.
• LC light chain
• NGHC non-glycosylated heavy chain
• LMW low molecular weight
• HMW high molecular weight

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